Method for inhibition of necrosis induced by neurotrophin

ABSTRACT

Disclosed is a method for inhibition of necrosis induced by neurotrophin, and more specifically a method for inhibition of necrosis by administrating oxidative stress inhibitor and a method for simultaneous inhibition of necrosis and apoptosis by administrating oxidative stress inhibitor and neurotrophin. The oxidative stress inhibitor of the present invention can inhibit nerve cell necrosis induced by neurotrophin. Moreover, it can be used for protecting nerve cell damage connected with alzheimer disease, parkinson&#39;s disease, degenerating cerebropathia and promoting regeneration of the nerve cells by administrating oxidative stress inhibitor and neurotrophin.

TECHNICAL FIELD

The present invention relates to a pharmacological composition forprevention of neuronal necrosis induced by neurotrophins, moreparticularity, to a method for prevention of neurotrophin-inducedneuronal death by anti-oxidants and synergetic effects of neurotrophinsand anti-oxidants for enhanced promotion of neuronal survival.

BACKGROUND ART

Survival of central and peripheral neurons largely depends upon contactwith neurotrophins that are released from their target cells(Levi-Montalcini, 1987, EMBO J., 6, 1145-1154; Barde, 1994, Prog. Clin.Biol. Res., 390, 45-56). The neurotrophic effect of neurotrophins isinitiated through binding to TrkA, TrkB, or TrkC, the high affinityneurotrophin receptors with tyrosine kinase activity (Patapoutian andReichardt, 2001, Curr. Opin. Neurobiol., 11, 272-280; Kaplan and Miller,2000, Curr. Opin. Neurobiol., 10, 381-391). The Trk tyrosine kinasesactivate the small GTP-binding protein Ras, PI-3K, and PLC, which playan important role in survival of a variety of neurons includingcerebellar granule, cortical, hippocampal, sympathetic, and sensoryneurons (Borasio et al., 1993, J. Cell Biol., 121, 665-672; Stephens etal., 1994, Neuron, 12, 691-705; Yao and Cooper, 1995, Science., 267,2003-2006; Nobes et al., 1996, Neuroscience., 70, 1067-1079; Nonomura etal., 1996, Brain Res Dev Brain Res., 97, 42-50; Alcantara et al., 1997,J Neurosci., 17(10), 3623-3633; Hetman et al., 1999, J Biol Chem., 274,22569-22580; Atwal et al., 2000, Neuron., 27, 265-227).

Neurotrophins enhance neuronal survival by interfering with programmedcell death or apoptosis in the process of normal development (Barde,1994, Prog. Clin. Biol. Res., 390, 45-56; Deshmukh and Johnson, 1997,Mol. Pharmacol., 51, 897-906). The neuroprotective effects ofneurotrophins have been observed in the central neurons subjected topathological insults. For example, neurotrophins ameliorate degenerationof basal forebrain cholinergic neurons, retinal ganglion neurons, andspinal sensory and motor neurons following axotomy in vivo (Hefti, 1986,J Neurosci., 6, 2155-2162; Yan et al., 1993, J. Neurobiol., 24,1555-1577; Mey and Thanos, 1993, Brain Res., 602, 304-317; Morse et al.,1993, J. Neurosci., 13, 4146-4156; Cohen et al., 1994, J. Neurobiol.,25, 953-959; Friedman et al., 1995, J. Neurosci., 15, 1044-1056). Nervegrowth factor (NGF), brain-derived neurotrophic factor (BDNF), andneurotrophins (NT)-4/5 can reduce neuronal death followinghypoxic-ischemic injury (Hefti, 1986, J. Neurosci., 6, 2155-2162; Yan etal., 1993, J. Neurobiol., 24, 1555-1577; Mey and Thanos, 1993, BrainRes., 602, 304-317; Morse et al., 1993, J. Neurosci., 13, 4146-4156;Cohen et al., 1994, J. Neurobiol., 25, 953-959; Friedman et al., 1995,J. Neurosci., 15, 1044-1056). BDNF protects dopaminergic neurons from1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 6-hydroxy dopamine(Spina et al., 1992, J. Neurochem., 59, 99-106; Frim et al., 1994, Proc.Natl. Acad. Sci. U.S.A., 91, 5104-5108).

The findings above suggest therapeutic potential of neurotrophins forhypoxic-ischemia and various neurodegenerative diseases. However, thebeneficial effects of neurotrophins should be compromised with a notionthat neurotrophins can exacerbate certain forms of neuronal injury.BDNF, NT-3, or NT-4/5 renders neurons highly vulnerable to deprivationof oxygen and glucose, possibly by enhancing Ca²⁺ influx and NO (nitricoxide) production through N-methyl-D-aspartate (NMDA) glutamatereceptors (Fernandez-Sanchez and Novelli, 1993, FEBS Lett., 335,124-131; Koh et al., 1995, Science, 268, 573-575; Samdani et al., 1997,J. Neurosci., 17, 4633-4641). BDNF, NGF, and NT-4/5 potentiate neuronalcell necrosis induced by oxidative stress or zinc in cortical cellcultures (Gwag et al., 1995, Neuroreport, 7, 93-96; Park et al., 1998,Neuroreport, 9, 687-690; Won et al., 2000, Neurobiol Dis, 7, 251-259).

Recently, the present inventors have found that neurotrophins candirectly induce neuronal cell necrosis in cortical cell cultures andadult rats as well as the potentiation effects of certain neuronalinjury (Kim et al., 2002, J. Cell Biol, 159, 821-831). Accordingly, theunexpected neurotoxicity of neurotrophins likely explains failure ofclinical trials in neuropathic pain and amyotrophic lateral sclerosis(Apfel et al., 2001, Clin. Chem. Lab. Med, 39(4), 351-61).

Thus, the inventors have delineated mechanisms underlying toxic effectsof neurotrophins, investigated drugs for prevention of neurotrophintoxicity, and completed the present invention by developing a method foroptimizing therapeutic effects of neurotrophins with anti-oxidants.

DISCLOSURE OF THE INVENTION

The present invention provides a method for preventingneurotrophin-induced neuronal cell necrosis with administration ofanti-oxidants, and also provides a method for preventing neuronalapoptosis and necrosis with concurrent administration of neurotrophinsand anti-oxidants.

The present invention provides a method for preventingneurotrophin-induced neuronal cell necrosis with administration ofanti-oxidants.

The present invention provides a method for preventing neuronalapoptosis and necrosis at the same time with concurrent administrationof neurotrophins and anti-oxidants.

The present invention provides a method for preventingneurotrophin-induced neuronal cell necrosis with administration oftetrafluorobenzyl derivatives.

The present invention provides a method for preventing neuronalapoptosis and necrosis with concurrent administration of neurotrophinsand tetrafluorobenzyl derivatives.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings.

FIG. 1 is a graph showing neurotrophins-induced neuronal necrosis incortical cell cultures.

-   -   A: treatment with BDNF B: treatment with NT-3 C: treatment with        NT-4/5

FIG. 2 is graph showing neuronal necrosis in brain sections stained withhematoxylin-eosin (H&E) at 2 day after intrastriatal injections ofsaline or BDNF.

A: Bright field photomicrogrphs of brain sections stained with H&E afterintrastriatal injections of saline

B: Bright field photomicrogrphs of brain sections stained with H&E afterintrastriatal injections of BDNF

C: a graph showing quantitative analysis of degenerating neurons inbrain sections stained with H&E after injections of saline or BDNF

FIG. 3 is a graph showing photomicrograph of cortical neurons 32 hrafter a sham wash or exposure to BDNF

A: Phase contrast photomicrograph of a sham wash

B: Phase contrast photomicrograph of BDNF

C: Electron photomicrograph of a sham wash

D: Electron photomicrograph of BDNF

FIG. 4 is a graph showing patterns of BDNF-induced neuronal death,degenerating neurons were defined as normal, necrosis (see above), orapoptosis, from sham wash group and control group at 32 hr followingexposure of cortical cell cultures to BDNF.

FIG. 5 is a graph showing neuronal death analyzed by measurement of LDHefflux into the bathing medium in cortical neurons after continuousexposure to BDNF, alone or with anti-BDNF blocking antibody, trolox, orCHX (cycloheximide).

FIG. 6 is a graph showing levels of ROS exposed to a sham wash or BDNFin cortical neurons analyzed at indicated times by measuringfluorescence intensity of oxidized DCDHF-DA (DCF).

FIG. 7 is a graph showing fluorescence quantitation of DCF in corticalneurons after 32 hr exposure of cortical cell cultures to a sham wash or100 ng/ml BDNF, alone or in the presence of CHX or trolox.

FIG. 8 is a graph showing RT-PCR analysis of NADPH oxidase and GAPDHmRNA expression in cortical cell cultures exposed to BDNF for indicatedtimes.

FIG. 9 is a graph quantitatively showing the mRNA level of NADPH oxidaseand GAPDH mRNA expression in cortical cell cultures exposed to BDNF forindicated times.

FIG. 10 is a graph showing western blot analysis of NADPH oxidase andactin expression in cortical cell cultures following exposure to BDNFfor indicated times.

FIG. 11 is a graph showing fluorescence photomicrograph of cortical cellcultures immunolabeled after exposure to a sham wash and BDNF.

A: sham wash, immunolabeling with anti-p47-phox or anti-goat IgGconjugated with FITC

B: sham wash, immunolabeling with NeuN or anti-goat IgG conjugated withTexas red

C: Treatment with BDNF, immunolabeling with anti-p47-phox or anti-goatIgG conjugated with FITC

D: Treatment with BDNF, immunolabeling with NeuN or anti-goat IgGconjugated with Texas red

FIG. 12 is a graph showing fluorescence photomicrograph of cortical cellcultures immunolabeled after exposure to a sham wash and BDNF.

A: sham wash, immunolabeling with anti-p67-phox or anti-goat IgGconjugated with FITC

B: sham wash, immunolabeling with NeuN or anti-goat IgG conjugated withTexas red

C: Treatment with BDNF, immunolabeling with anti-p67-phox or anti-goatIgG conjugated with FITC

D: Treatment with BDNF, immunolabeling with NeuN or anti-goat IgGconjugated with Texas red

FIG. 13 is a graph showing western blot analysis of the cytosolicfraction (C) and the membrane fraction (M) using anti-p47-phox andanti-p67-phox antibodies that were obtained from cortical cell culturesfollowing exposure to BDNF for indicated times.

FIG. 14 is a graph showing analysis of superoxide production bymeasuring reduction of cytochrome c in cortical cultures exposed to asham wash or BDNF with or without DPI for indicated times.

FIG. 15 is a graph showing fluorescence photomicrograph of the oxidizedHet and DCF in cortical neurons following exposure to a sham operation,BDNF, or BDNF plus DPI.

A: The oxidized hydroethydine (HEt) in cortical neurons followingexposure to a sham operation

B: The oxidized hydroethydine (HEt) in cortical neurons followingexposure to BDNF

C: The oxidized hydroethydine (HEt) in cortical neurons followingexposure to BDNF plus DPI

D: The oxidized DCF in cortical neurons following exposure to a shamoperation

E: The oxidized DCF in cortical neurons following exposure to BDNF

F: The oxidized DCF in cortical neurons following exposure to BDNF plusDPI

FIG. 16 is a graph showing analysis of neuronal death by measurement ofLDH efflux into the bathing medium in cortical neurons followingexposure to BDNF, BDNF+DPI, BDNF+AEBSF or BDNF+2-Hydroxy-TTBA.

FIG. 17 is a graph showing analysis of neuronal apoptosis in neuron-richcortical cell cultures following exposure to serum deprivation, alone orin the presence of BDNF, BDNF plus DPI, DPI, BDNF plus trolox, ortrolox.

DETAILED DESCRIPTION OF THE INVENTION

The detailed description of the present invention is as follows:

The present invention provides a method for preventingneurotrophin-induced necrosis with administration of drugs that blockoxidative stress.

Anti-oxidants in the present invention can be chosen from NADPH oxidaseinhibitors, vitamin E, vitamin E analogue or tetrafluorobenzylderivatives. NADPH oxidase inhibitors can be selected from diphenyleneiodonium (DPI) or 4-(2-amonoethyl)-benzensulfonyl fluoride (AEBSF).Vitamin E analogue is trolox, a membrane-permeable form of vitamin E.Tetrafluorobenzyl derivatives can be selected fromBAS(5-benzylaminosalicylic acid), TBAS(5-(4-trifluoromethylbenzyl)aminosalicylic acid), NBAS(5-(4nitrobenzyl) aminosalicylic acid),CBAS(5-(4-chlorobenzyl) aminosalicylic acid), MBAS(5-4-methoxybenzyl)aminosalicylic acid), FBAS(5-(4-fluorobenxyl) aminosalicylic acid), and2-hydroxy-TTBA(2-Hydroxy-5-(2,3,5,6-tetrafluoro-4trifluoromethyl-benzylamino)-benzoicacid.

Neurotrophins in the present invention can be selected from nerve growthfactor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3(NT-3), and NT-4/5, and BNDF is more preferred.

BDNF causes neuronal cell necrosis by inducing expression and activationof NADPH oxidase and subsequent production of reactive oxygen species(ROS).

Administration of DPI or AEBSF prevents BDNF-induced neuronal cellnecrosis by inhibiting NADPH oxidase and ROS production. Vitamin E or itanalogue trolox prevents BDNF-induced neuronal death by blocking ROSproduction. Tetrafluorobenzyl derivatives—BAS, TBAS, NBAS, CBAS, MBAS,FBAS, and 2-Hydroxy-TTBA—block free radical neurotoxicity asanti-oxidants (WO 01/79153), which prevents BDNF-induced neuronal death.

Thus, anti-oxidants in the present invention can preventneurotrophin-induced neuronal cell necrosis.

The present invention provides a method for preventing neuronalapoptosis and necrosis with concurrent administration of neurotrophinsand anti-oxidants.

Anti-oxidants in the present invention can be chosen from NADPH oxidaseinhibitors, vitamin E, vitamin E analogue or tetrafluorobenzylderivatives. NADPH oxidase inhibitors can be selected from DPI or AEBSF.Vitamin E analogue is preferably trolox, a membrane-permeable form ofvitamin E. Tetrafluorobenzyl derivatives can be selected from BAS, TBAS,NBAS, CBAS, MBAS, FBAS, and 2-Hydroxy-TTBA.

Neurotrophins in the present invention can be selected frombrain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), andNT-4/5, and BDNF is more preferred.

While neurotrophins promote neuronal survival by blocking apoptosis butcan cause neuronal necrosis through production of ROS. The latter can beblocked by administration of anti-oxidants. Interestingly, concurrentadministration of anti-oxidants greatly enhances effects ofneurotrophins promoting neuronal survival by blocking the pro-necroticactions of neurotrophins.

Thus, co-administration of neurotrophins and anti-oxidants can beapplied to prevent apoptosis and necrosis in hypoxic-ischemic injury(Holtzman et al., 1996, Ann. Neurol., 39(1), 114-122; Ferrer et al.,2001, Acta neuropathol.(Berl.), 101(3), 229-38), chronic spinal cordinjury (Jin et al., 2002, Exp. Neurol., 177(1), 265-75), Alzheimer'sdisease (Siegel and Chauhan, 2000, Brain Res. Brain Res. Rev., 33, 2-3),Parkinson's disease (Bradford et al., 1999, Adv. Neruol. 80, 19025), ALS(Louvel et al., 1997, Trends. Pharmacol. Sci., 18(6), 196-203),Huntington's disease (Perez-Navarro et al., 2000, J. Neurochem,75(5),2190-9), glaucoma (Ko et al., 2000, Invest. Ophthalmol. Vis. Sci.,41(10), 2967-71) or retinal detachment (Lewis et al., 1999, Invest.Ophthalmol. Vis. Sci., 40(7), 1530-1544).

The present invention provides an inhibitor for preventingneurotrophin-induced neuronal cell necrosis and thus enhancing survivaleffects of neurotrophins with administration of tetrafluorobenzylderivatives.

A drug containing tetrafluorobenzyl derivatives as an effectivecomponent can be applied to prevent ROS production and neuronal cellnecrosis by neurotrophins. Tetrafluorobenzyl derivatives in the presentinvention can be selected from BAS, TBAS, NBAS, CBAS, MBAS, FBAS, and2-Hydroxy-TTBA. Neurotrophins in the present invention can be selectedfrom brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3),and NT-4/5, and BDNF is more preferred.

The composition of the present invention can be treated by oraladministration, intravenous injection or non-oral administration, andtreated by various forms such as tablet, capsule, powder, grain,sterilized solution, suspension or suppository for rectaladministration. Major effective elements of the composition can be madeas a solid tablet using pharmaceutical carriers, for example commontablet element such as corn dextrin, lactose, sucrose, sorbitol, talc,stearic acid, magnesium stearate, decalcium phosphate or gums, andadditional pharmaceutical diluted solution. Tablets or pillets of thepharmaceutical composition in the present invention can be manufacturedfor sustained release dosage form as facilitated forms foradministration using well-known coating method etc. in the appropriateindustry. For example, tablets or pillets can be composed with inner andouter administrative elements. The inner administrative elements oftablets or pillets can be manufactured as wrapped with outeradministrative elements. Liquid forms of the composition in the presentinvention manufactured for oral administration or the injection includesolution, appropriately flavored syrup, water-soluble suspension,water-insoluble suspension, emulsion made by edible oil such as cottonoil, sesame oil, coconut oil, or peanut oil, elixir, and similarpharmaceutical carriers. Tragacanth gum, acacia, alginic acid sodiumsalt, dextran, sodium carboxymethylcellulose, methylcellulose,polyvinylpyrrolidone, or synthesized or natural gums like gelatin etccan be used as appropriated aid to dispersion or suspension in makingwater-soluble suspension.

Quantity of medication can be determined by several related factors suchas diseases, age, sex, weight, and degrees of illness of patients etc.for the treatment of neurodegeneration.

Hereinafter, embodiments of the present invention will be described indetail.

However, the examples described in the schemes are just representativeof the present invention, which could include more examples.

EXAMPLE 1 Primary Cortical Cell Cultures

Rat cortical cell cultures were prepared from the 17-day-old fetal brainand the neocortices were mechanically triturated as previously described(Noh and Gwag, 1997). Dissociated cells were plated on 6-well plates and24-well plates (approximately 3 cortices per plate), or on glass bottom35 mm dishes for ROS imaging. Plating media consist of Eagle's minimalessential media (MEM, Earle's salts, supplied glutamine-free)supplemented with 5% horse serum, 5% fetal bovine serum, 21 mM glucose,26.5 mM bicarbonate and 2 mM L-glutamine.

For neuron-glia mixed cultures, 10 μM cytosine arabinoside (Ara C) wasincluded to stop the overgrowth of non-neuronal cells to cultures at DIV5-7 when glial cells were confluent underneath neurons. After 2 days,cultures were then fed with plating medium lacking fetal serum twice aweek. Cultures were maintained at 37° C. in a humidified 5% CO₂incubator. For neuron-rich cultures (>95%/o), 2.5 μM Ara C was includedto cultures at 2-3 days in vitro (DIV 2-3) as previously described (Gwaget al., 1997).

EXAMPLE 2 Induction and Analysis of Cell Death

To examine if neurotrophins would induce neuronal necrosis, BDNF-inducedneuronal death was analyzed by measuring the level of lactatedehydrogenase (LDH) released into the bathing medium.

Mixed cortical cell cultures (DIV 12-14) were rinsed in serum free MS(MEM supplemented with 26.5 mM sodium bicarbonate and 21 mM glucose) andthen exposed to various concentrations of NGF, BDNF or NT-3 in serumfree MS. Neuronal cell death was analyzed by measuring the level oflactate dehydrogenase (LDH) released into the bathing medium. Thepercent neuronal death was normalized to the mean LDH value releasedafter a sham control (defined as 0%) or continuous exposure to 500 μMNMDA for 24 hr (defined as 100%) The latter produces complete neuronaldeath within 24 hr. For experiments for serum deprivation, neuron-richcortical cell cultures (DIV 7) were placed into serum free MS containing1 μM MK-801 as described (Gwag et al., 1995). Neuronal death wasanalyzed 24 and 48 hr later by counting viable neurons excluding TrypanBlue satained.

Wide spread neuronal death occurred in cortical cell culturescontinuously exposed to various concentrations (10, 30, or 100 ng/ml) ofBDNF or NT-3 for 36-48 hr (FIG. 1A). Near complete neuronal death wasobserved within 48 hr after exposure to BDNF or NT-3 (FIG. 1). Neuronalcell death was assessed by measurement of LDH efflux to the bathingmedium, mean±SEM (n=16 culture wells per condition). *, Significantdifference from the relevant control (sham washed control), at P<0.05using analysis of variance and Student-Newman-Keuls test.

Thus, it is found that neurotrophin such as BDNF, NT-3 and NT-4/5derives cell necrosis.

EXAMPLE 3 Intrastriatal Injection of BDNF in Rat Brain

To confirm BDNF-induced neuronal necrosis, adult male Sprague-Dawleyrats weighing 250-300 g were anesthetized intraperitoneally with chloralhydrate (400 mg/ml). Animals were placed in a Kopf stereotaxic apparatusand injected with 1 g/l of BDNF (dissolved in 0.9% NaCl (saline)), orsaline alone in the striatum at the following coordinates: 1.0 mmrostral to bregma, 3.0 mm lateral to the midline, and 5.0 mm ventralfrom the dural surface. For each injection, a volume of 5 μl wasdelivered for 10 min via 10 μl Hamilton syringe. Three minutes wereallowed prior to syringe withdrawal and wound closure. These rats wereeuthanized 2 d later. Animals were anaesthetized, and then perfusedtranscardially with phosphate-buffered saline (PBS) followed by 3%paraformaldehyde. The brains were immediately removed, post-fixed, andthen sectioned (8 μm) on a microtome (TPI, Inc., MO). Sections includingthe injection site were collected and stained with Hematoxylin and Eosin(H&E). The lesion area was analyzed as previously described (Won et al.,2000). Six serial sections including the needle track and the largestinjury area evident by decrease in staining intensity were included foranalysis of injury per each animal.

The striatal section stained with H&E was scanned analyzed using acomputer-assisted image analysis system (SigmaScan, Calif./TINA 2.0,KAIST, Daejeon, Korea). The neurotoxic effects of NTs were observed instriatal areas 2 d after the intrastriatal injections of BDNF in adultrat brain (FIG. 2).

EXAMPLE 4 Transmission Electron Microscopic Observation

To examine the patterns of BDNF-induced neuronal death, we observedneurons from 32 hrs after a sham wash or continuous exposure to 100ng/ml BDNF under phase contrast or transmission electron microscope.

Cultures were fixed in Karnovskys fixative solution (1%paraformaldehyde, 2% glutaraldehyde, 2 mM calcium cholride, 100 mMcacodylate buffer, pH 7.4) for 2 hr, washed with cacodylate buffer, andpost-fixed in 1% osmium tetroxide and 1.5% potassium ferrocyanide for 1hr. Cells were then stained en bloc in 0.5% uranyl acetate, dehydratedthrough graded ethanol series, and embedded in Poly/Bed 812 resin(Pelco, Calif.). Cells were sectioned using Reichert Jung Ultracut S(Leica, Cambridge, UK). After staining cells with uranyl acetate andlead citrate, cells were observed and photographed under Zeiss EM 902A.

The ultrastructural analysis of degenerating neurons in BDNF-treatedcortical cultures reveals swelling of cytoplasmic organelles, earliercollapse of plasma membrane than nuclear membrane, and scatteringcondensation of nuclear chromatin (FIG. 3 and 4). Neurons from controland BDNF-treated cultures were selected and observed under transmissionelectron microscope and degenerating neurons were defined as normal,necrosis, or apoptosis (shinkage of cytoplasm and nuclear membranerupture with intact plasma membrane), suggesting that BDNF inducedneuronal necrosis.

EXAMPLE 5 BDNF Produces ROS in Cortical Neurons

Many studies imply that increased reactive oxygen species (ROS) inducedneuronal necrosis, we examined if BDNF-induced neuronal necrosis wouldproduce ROS.

Cortical cell cultures (DIV 12-15) grown on glass bottom dishes wereloaded with 10 μM dichlorodihydro fluorescein diacetate (DCDHF-DA) or 5μM hydroethidium (Molecular Probes, Eugene, Oreg.) plus 2% PluronicF-127 in HEPES-buffered control salt solution (HCSS) buffer containing(in mM): 120 NaCl, 5 KCl, 1.6 MgCl₂, 2.3 CaCl₂, 15 glucose, 20 HEPES,and 10 NaOH. Cultures were incubated for 20 min at 37° C., and washedthree times with HCSS buffer. The fluorescence signal of oxidized DCDHFwas observed at room temperature on the stage of a Nikon Diaphotinverted microscope equipped with a 100 W xenon lamp and filter (foroxidized DCDHF, excitation=488 nm and emission=510 nm; forhydroethidine, excitation=546 nm and emission=590 mm). The fluorescenceimages were analyzed using a QuantiCell 700 system (Applied imaging,UK).

The fluorescent intensity of DCF was increased in cortical neuronsexposed to BDNF for 16 hr (FIG. 6). The intraneuronal levels of ROS([ROS]i) were further increased over 24-32 hr. The BDNF-inducedproduction of [ROS]_(i) was prevented by concurrent addition ofcycloheximide as well as trolox (FIG. 7).

Thus, BDNF likely produces ROS presumably through synthesis ofpro-oxidant proteins.

EXAMPLE 6 BDNF-Induced Neuronal Necrotic Mechanism

To examine BDNF-induced neuronal necrotic mechanism, cortical cellcultures (DIV 12-15) were continuously exposed to 100 ng/ml BDNF, aloneor with 100 μg/mi anti-BDNF blocking antibody, 100 μM trolox, or 1 μg/mlcycloheximide (CHX, and neuronal death was analyzed 36 hr later bymeasurement of LDH efflux into the bathing medium. Concurrentadministration of anti-BDNF blocking antibody completely blocked BDNFneurotoxicity.

Interestingly, BDNF-induced neuronal cell necrosis was also blocked byaddition of cycloheximide, a protein synthesis inhibitor and trolox, alipophilic analogue of vitamin E (FIG. 5). {mean±SEM (n=16 culture wellsper condition). *, Significant difference from the relevant control(BDNF alone), at P<0.05 using analysis of variance andStudent-Newman-Keuls test.}

Thus, neuroprotective effect against BDNF-induced neuronal necrosis wasaccompanied by blockade of reactive oxygen species (ROS) production.

EXAMPLE 7 Genes Expressed in Rat Cortical Cell Cultures Treated withBDNF

(7-1) cDNA Microarray Analysis

We used cDNA microarray assay to screen target genes for the pro-oxidantaction of BDNF in cortical cell cultures.

Total RNA was isolated from cortical cell cultures (DIV 12) by using RNAzol B (Tel-Test INC., Friendswood, Tex.). Approximately 1 μg of totalRNA was used to synthesize cDNA labeled with [α-³³p] dATP that washybridized to rat gene filter membranes (Research Genetics, Huntsville,Ala.) at 42° C. for 12-18 hr. The membranes were washed in 2× salinesodium citrate (SSC) buffer and 1% sodium dodecyl sulfate (SDS) at 50°C. for 20 min, 0.5×SSC and 1% SDS at room temperature for 15 min, andthen wrapped up in plastic wrap and apposed to a phosphorimagercassette. After exposure of gene filters, the hybridization pattern wasanalyzed using Pathways™ 4-universal microarray analysis softwareInvitrogen, Netherlands).

The microarray analysis revealed that various genes were regulated incortical cell cultures exposed to BDNF for 8 hr (Table 1). The targetgenes of BDNF mostly play a role in differentiation, endocytosis,metabolism, and signal transduction that likely reflect neurotrophicactions of neurotrophins. Among the BDNF-sensitive genes, cytochromeb₅₅₈ was chosen as a candidate gene for the neurotoxic actions of theNTs, since it constitutes p22-phox and gp91-phox subunits of NADPHoxidase, a pro-oxidant enzyme generating superoxide from oxygen.

(7-2) Reverse Transcription—Polymerase Chain Reaction (RT-PCR)

RT-PCR experiments were performed to confirm the BDNF-sensitive genesderived from cDNA expression microarray, cytochrome b₅₅₈ as itconstitutes p22-phox and gp91-phox subunits of NADPH oxidase, apro-oxidant enzyme generating superoxide from oxygen.

Total RNA (1 μg each) was incubated in a reaction mixture containingdNTP (2.5 mM each), RNasin (0.5 Unit), oligo dT primer (100 ng), andMMLV reverse transcriptase (200 Unit) at 37° C. for 1 hr. The sampleswere incubated at 92° C. for 10 min and transferred to 4° C. The reversetranscribed cDNA was subjected to PCR amplification. PCR was performedaccording to manufacturer's procedure (Takara Shuzo Co., Japan)sequentially (denaturation-annealing-extension) at following conditions:for p47-phox, 94° C. for 30 S, 55° C. for 30 S, and 72° C. for 60 S (28cycles); for p22-phox (homologous to cytochrome b₅₅s₈ in microarray) andgp91-phox, 94° C. for 45 S, 60° C. for 60 S, and 72° C. for 120 S (33cycles); for GAPDH, 94° C. for 35 S, 55° C. for 45 S, and 72° C. for 90S (25 cycles). Primer sequences used were as follows (5′-3′): forp22-phox, GAATTCCGATGGGGCAGATCGAGTGGGCCA (forward) and GGATCCCGTCACACGACCTCATCTGTCACT (reverse); for p47-phox, CAGCCA GCACTATGTGTACA(forward) and GAACTCGTAGATCTCGGTGAA (reverse); for gp91-phox,GAATTCCGATGGGGAACTGGGCTGTGAA TG (forward) andGGATCCCGTTAGAAGTTTTCCTTGTTGAAA (reverse); for GAPDH,TCCATGACAACTTTGGCATCGTGG (forward) and GTTGCTGTTGAAGTCACAGGAGAC(reverse). PCR products were run on a 1.2% agarose gel and visualizedwith ethidium bromide. The relative amount of mRNA was measured usingLAS-1000 systems (Fuji Photofilm Co., Japan), normalized to levels ofGAPDH mRNA. DNA sequencing was performed with Big Dye TerminatorChemistry from Perkin-Elmer Applied Biosystems on ABI PRISM™ 377 DNAsequencer (Foster City, Calif.)

Reverse transcription-polymerase chain reaction (RT-PCR) analysis showedincrease in mRNA levels of p22-phox and gp91-phox within 2 hr aftertreatment with BDNF. Levels of both mRNAs were maximally increased 4 hrlater, which lasted over the next 12 hr. The mRNA levels of p47-phoxsubunit were also increased gradually from 30 min followingadministration of BDNF (FIG. 8 and 9).

Thus, BDNF appears to increase expression of NADPH oxidase.

(7-3) Western Blot Analysis

Western blot experiments were performed to analyze protein levels ofNADPH oxidase subunits using available antibodies for gp91-phox,p47-phox, and p67-phox.

Cortical cell cultures were lysed in a lysis buffer containing 50 mMTris-HCl (pH 7.5), 1% Nonidet P-40, 150 mM NaCl, 0.5% deoxycholic acid,0.1% sodium dodecyl sulfate (SDS), 1 mM PMSF (phenylmethylsulfonylfluoride), 10 μg/ml pepstain A, and 100 μg/ml leupeptin. Cell lysateswere centrifuged at 12,000 g for 10 min. approximately 25 μg of proteinwas subjected to electrophoresis on 12% SDS-polyacrylamide gel andtransferred to a nitrocellulose membrane. The blot was incubated in 2.5%bovine serum albumin for 1 hr, incubated with goat polyclonal primaryantibodies, anti-gp91-phox, anti-p67-phox, or anti-p47-phox antibodies(1:1000, Santa Cruz, Santa Cruz, Calif.), and then reacted with abiotinylated anti-goat secondary antibody. Immunoreactivity was detectedwith Vectastain ABC kit (Vector Laboratory, Burlingame, Calif., USA) andluminol for enhanced chemiluminescence Intron, Korea). The signal wasanalyzed by quantitative densitometry using LAS-1000 systems (FujiPhotofilm Co., Japan).

Protein levels of NADPH oxidase subunits, gp91-phox, p47-phox, andp67-phox, was increased over 16-32 hr in cortical cell cultures exposedto BDNF (FIG. 10).

Thus, BDNF appears to express protein levels of NADPH oxidase.

(7-4) Immunocytochemistry

Immunocytochemistry was performed to identify which types of cellsexpress NADPH oxidase in cortical cell cultures containing neurons andglia.

Cortical cell cultures (DIV 12-14) grown on glass bottom dishes werefixed in 4% paraformaldehyde for 30 min, incubated in 10% horse serumfor 1 hr, and double-immunolabeled with a mouse monoclonal antibodyagainst NeuN (1:400 dilution, Chemicon, Temecula, Calif.) and a goatpolyclonal antibody against p47-phox or p67-phox (1:200 dilution, SantaCruz, Santa Cruz, Calif.) for 2-4 hr. Cultures were then reacted withfluorescein isothiocyanate-conjugated anti-goat IgG (1:200 dilution,Organon Teknika Corp., NC) and Texas red-conjugated anti-mouse IgG(1:200, Vector Laboratory, Burlingame, Calif.) for 1-2 hr. Thefluorescence images were collected and analyzed with a fluorescencemicroscopy (Zeiss, Germany) equipped with the Real-14™ precision digitalcamera (Apogee Instrument, Tucson, Ariz.) and ImagePro Plus Plug-in(Silver Spring, Md.).

Immunoreactivity to p47-phox or p67-phox antibody was slightly observedin cortical neurons but not in astrocytes following a sham operation.Signals of p47-phox and p67-phox were increased exclusively in neurons32 hr following exposure of cortical cell cultures to BDNF (FIG. 11 and12).

(7-5) Subcellular Fractionation

Activation of NADPH oxidase involves translocation of the cytosolicp47-phox and p67-phox subunits into the plasma membrane (Clark et al.,1989, Trans. Assoc. Am. Physicians., 102, 224-230). We examined iftreatment with BDNF would activate NADPH oxidase through isolating thecytosol and membrane fraction.

Cortical cell cultures were washed with ice-cold PBS and resuspended inan isotonic buffer containing 10 mM HEPES, pH 8.0, 250 mM sucrose, 1 mMEDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT), 2 mM PMSF, 100 μg/mlleupeptin, and 10 μg/ml pepstatin A. For isolating the cytosol andmembrane fraction, the lysate was homogenized with a homogenizer (KONTE,Vieland, N.J.), centrifuged at 9,000 g for 10 min, and the supernatantwas then centrifuged at 100,000 g for 1 hr.

The membrane fraction was obtained by resuspending the pellet with 50 μllysis buffer and the cytosolic fraction was obtained from thesupernatant. As shown in <EXAMPLE 7-4>, western blot experiments wereperformed to analyze protein levels of NADPH oxidase subunits usingavailable antibodies for gp91-phox, p47-phox, and p67-phox.

The levels of p47-phox and p67-phox were reduced in the cytosolicfraction and increased in the membrane fraction from cortical cellcultures exposed to BDNF for 16-32 hr (FIG. 13), suggesting thattreatment with BDNF-induced activation of NADPH oxidase was increased inthe membrane fraction, and induced oxidative stress in cortical neuronsthrough production of ROS.

EXAMPLE 8 Measurement of NADPH Oxidase Activity in BDNF-Treated CoricalCell Cultures

We examined if activation of NADPH oxidase would contribute toBDNF-induced neuronal death.

Superoxide production was measured in a quantitative kinetic assay basedon the reduction of cytochrome c (Mayo and Curnutte, 1990). Corticalcell cultures were suspended in PBS and incubated in a reaction mixturecontaining 0.9 mM CaCl₂, 0.5. mM MgCl₂, and 7.5 mM glucose, 75 μMcytochrome c (Sigma, St. Louis, Mo.), and 60 μg/ml super oxide dismutase(Sigma, St. Louis, Mo.) for 3 min at 37° C. The superoxide productionwas determined by measuring the absorbance of cytochrome c at 550 nmusing a Thermomax microplate reader and associated SOFTMAX Version 2.02software (Molecular Devices Corp., Menlo Park, Calif.).

Co-administration of NADPH oxidase inhibitors, 3-10 nM DPI or 10-30 μM4-(2-amonoethyl)-benzensulfonyl fluoride (AEBSF), significantly reducedswelling of neuronal cell body and neuronal death 36 hr after exposureof cortical cell cultures to BDNF (FIG. 14).

Thus, the NADPH oxidase-mediated production of superoxide was reduced inthe presence of the selective inhibitors of NADPH oxidase.

EXAMPLE 9 BDNF Produces ROS Through Activation of NADPH Oxidase

NADPH oxidase was first discovered in phagocytes as asuperoxide-producing enzyme via one-electron reduction of oxygen. Weperformed to examine whether BDNF would produce ROS through activationof NADPH oxidase.

Superoxide production through activation of NADPH oxidase was analyzedby measuring the oxidation of HEt and DCDHF to ethidium and DCF,respectively, in cortical neurons after exposure to a sham operation,100 ng/ml BDNF, or 100 ng/ml BDNF plus 3 nM DPI for 32 hr (FIG. 15).Treatment with BDNF resulted in the increased oxidation of Het andDCDHF, DPI completely blocked BDNF-induced superoxide production (FIG.15).

Thus, BDNF produces oxidative stress in cortical neurons through NADPHoxidase-mediated production of superoxide.

EXAMPLE 10 Activation of NADPH Oxidase Mediates BDNF Neurotoxicity

We examined if activation of NADPH oxidase would contribute toBDNF-induced neuronal death.

Cortical cell cultures (DIV 12-15) were exposed to 100 ng/ml BDNF, aloneor in the presence NADPH oxidase inhibitors, 3-10 nM DPI, 10-30 μM4-(2-amonoethyl)-benzensulfonyl fluoride (AEBSF), or 1 μM 2-Hydroxy-TTBA((2-Hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethyl-benzylamino)-benzoicacid). Neuronal death was analyzed 36 hr later by measurement of LDHefflux into the bathing medium.

Co-administration of 3 nM DPI, 10 μM AEBSF, or 1 μM 2-Hydroxy-TTBAsignificantly reduced neuronal necrosis 36 hr after exposure of corticalcell cultures to BDNF (FIG. 16). Mean±SEM (n=16 culture wells percondition). *, Significant difference from the relevant control (BDNFalone), at P<0.05 by Student-Newman-Keuls test.

Thus, the neurotrophic effect of BDNF is enhanced with blockade ofoxidative stress by NADPH inhibitors or antioxidants.

EXAMPLE 11 Antiapoptotic Action of NTs

It has been reported that BDNF prevent neuronal apoptosis. As previouslyreported, we examined if blocking action of BDNF, such as inhibitor ofNADPH oxidase or antioxidant, would affect BDNF-induced neuronalnecrosis.

Neuron-rich cortical cell cultures (DIV 7) were deprived of serum, alone(serum deprivation) or in the presence of 100 ng/ml BDNF, 100 ng/ml BDNFplus 3 nM DPI, 3 nM DPI, 100 ng/ml BDNF plus 100 μM trolox, or 100 μMtrolox. Neuronal death was assessed 24 hr and 48 hr later by countingviable neurons.

Neither DPI nor trolox alone reduced serum deprivation-induced neuronalapoptosis. The anti-apoptotic action of BDNF was insensitive toinclusion of DPI or trolox. Interestingly, the protective effects ofBDNF disappeared within 48 hrs following serum deprivation. The delayedneuronal death evolving in the presence of BDNF attenuated by concurrentaddition of DPI or trolox (FIG. 17). Mean±SEM (n=16 fields randomlychosen from four culture wells per condition). *, Significant differencefrom the relevant control (serum deprivation alone), at P<0.05 usinganalysis of variance and Student-Neuman-Keuls test.

Administration of BDNF prevented neuronal apoptosis, but prolongedexposure to BDNF produces neuronal cell necrosis without blockinganti-apoptosis action of BDNF.

Above results show that BDNF-induced expression and activation of NADPHoxidase cause oxidative neuronal necrosis and that the neurotrophiceffects of NTs can be maximized under blockade of the pronecroticaction. The concrete diseases applicable with antioxidants or itsneurotrophins are described as follows.

Application examples described below are part of examples of thisinvention. This invention is not limited to application examples.

APPLICATION EXAMPLE 1 Alzheimer's Disease (AD)

The degeneration of glutamatergic neurons in the cerebral cortex andhippocampal formation and of cholinergic neurons in the basal forebrain,extracellular deposit of amyloid plaque, and intracellularneurofibrillary tangles are pathological features of AD. The ability ofneurotrophins (e.g., nerve growth factor [NGF]) to promote the survivaland phenotype of subsets of central nervous system (CNS) neuronsvulnerable in AD, such as basal forebrain cholinergic neurons suggeststhe use of these molecules to treat neurodegeneration associated withhuman diseases (Hefti, 1994, J Neurobiol., 25, 1418-1435). In AD, theproduction of lipid peroxidation, 8-hydroxy deoxyguanosine,protein-carbonyls, nitration, or oxidative crosslinking of proteins byexcess generation of free radicals has been reported, suggesting thatoxidative stress plays a causative role in neuronal death in AD [Viteket al., Proc. Natl. Acad Sci. U.S.A., 91:4766-4770 (1994); Smith et al.,Trends. Neurosci., 18:172-176 (1995), Mol. Chem. Neuropathol., 28:4148(1996), Proc. Natl. Acad Sci. U.S.A., 94:9866-9868 (1997); Montine etal., J. Neuropathol. Exp. Neurol., 55:202-210 (1996)]. As a matter offact, the therapeutic effects of anti-oxidants have been extensivelyinvestigated in AD patients. Zn²⁺ is accumulated in the brain (amygdala,hippocampus, inferior parietal lobule, superior and middle temporalgyri) of AD patients, mainly in the center and surround of amyloidplaque and induces aggregation of beta amyloid [Lovell et al., J.Neurol. Sci., 158:47-52 (1998)]. Therefore, the compounds in the presentinvention showing protective effect against oxidative stress and Zn²⁺toxicity can be used as therapeutic drugs for AD.

APPLICATION EXAMPLE 2 Parkinson's Disease (PD)

PD is a neurodegenerative disease showing the disorder of motor functionby a selective death of dopaminergic neurons in the substantia nigra. InPD patients, oxidative stress has been proved as a main mechanism ofneuronal cell death, and the production of lipid peroxidation, 8-hydroxydeoxyguanosine, protein carbonyls or nitration has been reported,suggesting that oxidative stress plays a causative role in neuronaldeath in PD (Tatton and Kish, 1997, Neuroscience, 77, 1037-1048; He etal., 2000, Brain Research, 858, 163-166; Turmel et al., 2001, Mov.Disord., 16, 185-189). Many in vivo studies have shown that there issome evidence for the occurrence of apoptosis in the parkinsoniansubstantia and neurotrophins such as BDNF or GDNF (Glial cell-derivedneurotrophic factor) also prevents the death of dopaminergic neurons invivo (Bradford et al., 1999, Adv. Nerurol., 80, 19-25; Levivier et al.,1995, J. Neurosci., 15, 7810-7820; Olson, 1996, Nat. Med, 2, 400-401;Gash et al., 1996, Nature, 380, 252-255).

Therefore, the compounds in the present invention showing neurotrophiceffects of NTs and antioxidant effects can be used as therapeutic drugfor PD.

APPLICATION EXAMPLE 3 Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurologicaldisorder that is characterized by the selective degeneration of lowerand/or upper motor neurons leading to progressive weakness, atrophy ofskeletal muscles and eventual paralysis and death within 2-5 years ofclinical onset. Autosomal-dominant familial ALS (FALS) have pointmutations in the gene that encodes Cu/Zn superoxide dismutase (SOD1),and protein carbonyl content, a marker of oxidative damage, was elevatedin the SALS patients relative to the control patients (Bowling et al.,1993, J. Neurochem., 61, 2322-2325). Recently, clinical trial of BDNFhas failed to show beneficial effects in amyotrophic lateral sclerosis(Apfel et al., 2001, Clin. Chem. Lab. Med, 39(4), 351-61). Thisunfavorable effects may be attributable to oxidative neuronal death bythe neurotrophin. Thus, the concurrent administration of neurotrophinsand anti-oxidants can be applied to effectively treat ALS.

APPLICATION EXAMPLE 4 Hypoxic-Ischemic Injury

Stroke occurs when local thrombosis, embolic particles, or the ruptureof blood vessels interrupts the blood flow to the brain. Duringhypoxic-ischemia, membrane depolarization triggers excess Ca²⁺ influx inneurons and glia, reflecting subsequent accumulation of Ca²⁺ inmitochondria ([Ca^(2+])m). Excess Ca²⁺ in the mitochondria results inthe production of flee radicals. Accumulated ROS in cells are expectedto randomly attack DNA, lipid, and protein; therefore, they contributeto hypoxic-ischemic neuronal necrosis. The pharmacological or geneticintervention of ROS and RNS has been neuroprotective againsthypoxic-ischemic neuronal necrosis. (Holtzman et al., 1996, Ann.Neurol., 39(1), 114-122; Ferrer et al., 2001, Acta neuropathol.(Berl.),101(3), 229-38; Hall et al., 1990, Stroke, 21, 11183-11187). As DNAladders, TUNEL-positive neurons and chromatin condensation were observedin the process of neuronal death in the hypoxic-ischemic brain areas;apoptosis, as well as necrosis, have been considered as additional typesof hypoxic-ischemic neuronal death. Thus, the concurrent administrationof neurotrophins and anti-oxidants can be applied to effectively treathypoxic-ischemic injury.

APPLICATION EXAMPLE 5 Chronic Spinal Cord Injury

Traumatic injuries to spinal cord cause tissue damage, in part byinitiating reative biochemical changes. Numerous studies have providedconsiderable support for lipid peroxidation reactions, Ca2+ influx, anddisruption of membrane in the spinal cord injury (Brown and Hall, 1992,J. Am. Vet. Med. Assoc., 200, 1849-1859; Springer et al., 1997, J.Neurochem., 68, 2469-2476; Juurlink and Paterson, 1998, J. Spinal cordMed., 21, 309-334), recent evidences provide that neuronal necrosis suchas, glutamate excitotoxicity, Ca2+ overload, and oxidative stress, alsocauses secondary damage, and that special Caspase 3 enzyme inhibitor canapparently decrease the neuronal apoptosis in brain trauma model (Zhanget al., 1990, J. Neurochem., 59, 733-739). Thus, the concurrentadministration of neurotrophins and anti-oxidants can be applied toeffectively treat chronic spinal cord injury.

APPLICATION EXAMPLE 6 Huntington's Disease (HD)

Striatal projection neurons are highly vulnerable to apoptosis in HD,and oxidative stress contributes to apoptosis of striatal projection,neurotrophic fators are protein that support the survival of neurons,maintain their functions and protect them from different types ofinsults. Recent reports have shown that grafting of the neurotrophinssuch as GDNF or BDNF-secreting cell line, protects striatal projectionneurons in a rat model of Huntington's disease (Perez-Navarro et al.,2000, J. Neurochem., 75(5), 2190-9). Therefore, the compounds in thepresent invention showing neuroprotective effects of coadministrationwith neurotrophins and antioxidants can be used as therapeutic drugs forHD.

APPLICATION EXAMPLE 7 Glaucoma and Retinal Detachment

Glaucoma is a chronic, progressive optic neuropathy often leading toblindness. Elevated intraocular pressure (IOP) is the most importantrisk factor for progression of glaucomatous damage. Death of retinalganglion cells (RGCs) in glaucomatous eyes occurs by apoptosis asdemonstrated in different species. Reduction of IOP remains the mostcommon treatment for glaucoma. Recent studies provide that free radicalscavengers and neurotrophins and other growth factors promote RGCsurvival and control damage induced by elevated IOP in animal models (Koet al., 2000, Invest. Ophthalmol. Vis. Sci., 41(10), 2967-71). Recentevidences provide that BDNF may aid in the recovery of the retina afterreattachment by maintaining the surviving photoreceptor cells, byreducing the gliotic effects in Muller cells, and perhaps by promotingouter segment regeneration (Lewis et al., 1999, Invest. Ophthalmol. Vis.Sci., 40(7), 1530-1544). Thus, the concurrent administration ofneurotrophins and anti-oxidants can be applied to effectively treatglaucoma and retinal detachment.

INDUSTRIAL APPLICABILITY

As anti-oxidants used in the present invention blockneurotrophin-induced neuronal necrosis without influencinganti-apoptosis actions of neurotrophins, concurrent administration ofanti-oxidants and neurotrophins can be applied to prevent neuronalinjury and to promote regeneration in acute brain diseases such asstroke and trauma as well as neurodegenerative diseases such asAlzheimer's disease and Parkinson's disease.

1. A method for prevention of neurotrophin-induced neuronal death, byadministration of anti-oxidants.
 2. A method for prevention ofneurotrophin-induced neuronal death according to claim 1, whereinneurotrophin is administered together with the anti-oxidants.
 3. Amethod for prevention of neurotrophin-induced neuronal death accordingto claim 1 or 2, wherein the neurotrophin is selected from the groupconsisting of nerve growth factor (NGF), brain-derived neurotrophicfactor (BDNF), neurotrophin-3 and NT4/5.
 4. A method for prevention ofneurotrophin-induced neuronal death according to claim 1 or 2, whereinthe neurotrophin is BDNF.
 5. A method for prevention ofneurotrophin-induced neuronal death according to claim 1 or 2, whereinthe anti-oxidant is selected from the group consisting of NADPH oxidaseinhibitors, vitamin E, vitamin E analogue and tetrafluorobenzylderivatives
 6. A method for prevention of neurotrophin-induced neuronaldeath according to claim 5, wherein the NADPH oxidase inhibitor is atleast one selected from the group consisting of diphenylene iodonium(DPI) and 4-(2-amonoethyl)-benzensulfonyl fluoride (AEBSF).
 7. A methodfor prevention of neurotrophin-induced neuronal death according to claim5, wherein the vitamin E analogue is trolox.
 8. A method for preventionof neurotrophin-induced neuronal death according to claim 5, wherein thetetrafluorobenzyl derivatives is at least one selected from the groupconsisting of BAS(5-benzylaminosalicylic acid),TBAS(5-(4-trifluoromethylbenzyl) aminosalicylic acid),NBAS(5-(4-nitrobenzyl) aminosalicylic acid), CBAS(5-(4-chlorobenzyl)aminosalicylic acid), MBAS(5-(4-methoxybenzyl) aminosalicylic acid),FBAS(5-(4-fluorobenxyl) aminosalicylic acid) and2-hydroxy-TTBA(2-Hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethyl-benzylamino)-benzoicacid).
 9. A method for prevention of neurotrophin-induced neuronal deathaccording to claim 2, wherein the method is used for therapy orprophylaxis of Hypoxic-ischemic injury, Chronic spinal cord injury,Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis,Huntington's disease, Glaucoma or Retinal detachment
 10. A method forprevention of neurotrophin-induced neuronal death according to claim 1or 2, wherein the neuronal death is neuronal apoptosis and/or necrosis.11. An inhibitor for neurotrophin-induced neuronal death, characterizedin that the inhibitor contains at least one selected from the groupconsisting of tetrafluorobenzyl derivatives including BAS, TBAS, NBAS,CBAS, MBAS, FBAS and 2-hydroxy-TTB, as an effective component
 12. Aninhibitor for neurotrophin-induced neuronal death according to claim 11,further comprising neurotrophin as an effective component.
 13. Aninhibitor for neurotrophin-induced neuronal death according to claim 11or 12, wherein the neuronal death is neuronal apoptosis and/or necrosis.